Broadly applicable, accurate, sensitive and automatable assays are needed to monitor the presence and quantity of biological materials present in complex body fluids of patients at micromolar to picomolar concentrations in order to aid in diagnosis and therapy of disease.
Various methods utilized in the past, including liquid and gas chromatography, mass spectrometry, and numerous bioassay techniques, are time-consuming, costly and not readily automated.
Competitive protein binding assays such as radioreceptor assays and radioimmunoassays provided a major improvement in analytical sensitivity and productivity, but have the disadvantages of dealing with hazardous radioactive materials, and not being amenable to automation. While enzyme-linked and chemiluminescence-linked immunoassays and DNA probe assays have eliminated the hazardous radioactive materials, they have not solved the problem of a lack of automatability.
In recent years, a number of particle-based immunoassays have been developed to take advantage of the specificity of antibody reactions, while avoiding the complications of radiochemical labelling. Agglutination reactions involving bivalent antibodies and antigens or haptens of clinical interest have been utilized in both visual and quantitative assays with a wide variety of bacteria, red blood cells or polymer particles. Agglutination results from the growth of antibody (Ab)-antigen (Ag)-bridged particle aggregates to produce an extensive network that can be detected. Agglutination can result by adding the specific binding partner, either Ab or Ag, to the suspension of particles with immobilized Ab or Ag. At low concentrations of the specific binding partner, small aggregates consisting of only a few particles are produced. Particle-based diagnostic tests are usually based upon the very specific interaction of Ag and Ab. Ab or Ag can be adsorbed on submicron-sized polystyrene particles, often called "uniform latex particles". Bangs, L. B., Uniform Latex Particles Indianapolis: Seragen, 1984. These sensitized particles then act to amplify the visibility of the Ab-Ag reaction that takes place when a sample containing the sought Ag or Ab is mixed with these appropriately coated particles.
Suspensions of polymer microparticles in the colloidal size range of 0.02-100 .mu.m diameter particles are available commercially. The properties of these particles are determined predominantly by the physicochemical properties of their surfaces. A single polystyrene latex particle is composed of a large number of individual polystyrene molecules (&gt;1000 even for a particle as small as 0.1 .mu.m diameter) held together by van der Waal's attractive forces. Each polymer molecule in the particle has end functional charge groups that are usually hydrophilic and that originate from a fragment of the compound used as the initiator of the polymerization. See, for a review, Seaman, G. V. F., "Physicochemical Properties of Latexes in Design of Latex Tests", in Seaman, G. V. F., ed., Applying Latex Based Technology in Diagnostics, Health & Science Commun., Washington, D.C., 1990, pp.1-19.
The usual form of polystyrene latex particles possesses sulfate charge groups for stabilization, but a variety of other functional groups can be introduced at the particle surface, such as hydroxyl, carboxyl, amine, and polymeric carboxylate groups. Such groups are particularly advantageous for binding to latex bead surfaces a wide variety of ligands and receptors.
Although, as noted above, sizes of polystyrene microspheres available commercially cover the range of 0.2 .mu.m up to about 100 .mu.m, the sizes used for serodiagnostic testing are predominantly in the range of 0.1 to 1.0 .mu.m diameter. Performance characteristics are all influenced by particle size and monodispersity. Sedimentation under the force only of gravity may occur with the larger diameter microspheres, although this is not a major problem in the size ranges used in agglutination assays.
Uncoated latex particles form relatively stable hydrophobic suspensions because of like charge on all microspheres. When coated with a ligand such as Ag or Ab, the particles form stable hydrophilic suspensions remaining dispersed during storage, but aggregating when reacted with a complementary cross-reacting anti-ligand. The ligands used to coat latex particles are attached by one of three methods: (i) physical (passive) absorption; (ii) facilitated (forced) absorption; and (iii) covalent coupling.
Latex agglutination tests can employ either agglutination or inhibition of agglutination of particles. Conventional agglutination tests are used for the detection of Ab's or relatively high molecular weight Ag's, while agglutination inhibition tests are used principally for the detection of low molecular weight Ag's and also some larger Ag's.
When optical instruments that measure transmitted, absorbed or scattered light are used, it is possible to estimate agglutination of coated latex particles quantitatively and to develop sensitive particle immunoassays. The intensity of light scattered by particles dispersed in water varies with the number of particles, their diameter, the wavelength of the incident light, the angle of the detector to the incident light and several other variables.
As agglutination starts, single particles first become doublets; the number of monomeric light-scattering particles drops dramatically, and the apparent diameter of agglutinates increases rapidly. After this point, the changes in numbers and diameters are less rapid. An important aspect of particle agglutination, disclosed in the present invention below, is that scattered light intensity measured as a function of time can be the basis for a very sensitive kinetic immunoassay.
Several methods for quantifying particle immunoassays have already been devised. Instruments such as Coulter Counters.RTM. (Coulter Electronics, Hialeah, Fla.) that count numbers of particles or clumps of particles in discrete channels have been used to follow agglutination. As the particles of small size agglutinate, the signals disappear from one channel and appear in higher channels. One can thus count single particles as they decrease in number or count clumps of newly aggregated particles as they increase.
One can use a nephelometer to follow scattered light directly or a spectrophotometer to measure change of "absorbance" of light (measure scattered light indirectly). Angular anisotropy or dynamic light scattering or photon correlation spectroscopy are newer, even more powerful techniques for measuring particle agglutination quantitatively.
As noted above, traditionally, optical instruments such as turbidimeters or nephelometers that rely upon light scattering differences between agglutinated and unagglutinated particles have been applied to the problem of quantitating latex particle agglutination tests. Although such methods preserve the advantage of monitoring a homogeneous reaction mixture without the need for a separation step, they are satisfactory only for single tests and are not satisfactory for simultaneous, quantitative, multiple latex particle agglutination tests, which is the subject of the present invention below.
Light scatter from bulk solution of aggregating or dissociating immunobeads can be used to provide quantitative measurements of analyte concentrations. Turbidimeters measure light transmission through a suspension of particle aggregates, and nephelometers directly measure scattered light in specific directions. In both instances, light scatter from a mixed population of both aggregated and nonaggregated particles is measured. See, e.g., the LPIA.RTM. nephelometer instrument of Mitsubishi Chemical Ind., Tokyo that is capable, however, of analyzing only one analyte at a time; Kapmeyer et al., U.S. Pat. No. 4,305,925 which discloses a nephelometric method wherein two different particle sizes are used to enhance the useful range of a latex agglutination immunoassay of a single analyte; and Ziege et al., WO 90/08961 who discloses a nephelometric quantitative immunoassay which employs coordinated carrier particles composed of copolymeric materials for the detection of a single analyte. There is no obvious way to extend the teachings of these patents to the use of a multiplicity of particle sizes to measure different analytes simultaneously.
It is well known that particles of different sizes, shapes and composition relative to the wavelength of light will scatter light differently in different directions. M. Kerker, The Scattering of Light and Other Electromagnetic Radiation, Academic Press, N.Y. 1969. It would be theoretically appealing to attempt to use the different angular scattering patterns of different particles in bulk solution in order to perform simultaneous assays. In practice, however, there is so much overlap in the angular scattering patterns of different particles that it becomes impossible to separate the results of one agglutination reaction from another.
As will be detailed below, the present invention employs an instrument for its simultaneous, quantitative multiple assay method that is neither turbidimetric nor nephelometric, but instead monitors light scattered from single particles or particle aggregates rather tan from many particles in bulk solution, and belongs to the class of instruments known as Flow Particle Analyzers (FPA).
Two types of FPAs have been used to detect particle aggregation by monitoring the size of individual particles or aggregates thereof as they flow individually through either an electronic or optical sensing zone. In the first type, particles and particle aggregates flow through a physically small, electronic sizing orifice, and in the second type the particles and aggregates flow through a focused optical beam. Although these approaches have been applied to quantitative latex particle agglutination assays (see below), neither has been successfully applied to the problem of simultaneous, quantitative multiple latex bead agglutination tests, and are limited to single tests or require complex signals to measure multiple analytes in a single sample.
Although electronic flow-through orifices can detect size differences among a population of electrically insulating particles, there are certain practical limits to using such devices in latex particle agglutination tests, due, in part, to the clogging of sensitive sizing orifices by high order aggregates unavoidably produced during agglutination tests and by particulate sample impurities. Masson, P. L. et al., Methods in Enzymology, 74:106 (1981); Cohen, R., U.S. Pat. No. 4,851,329. This limitation has prevented any routine, practical use of electronic sizing orifices in attempts to quantify latex particle agglutination tests.
As optical FPAs can use large bore capillary sensing chambers and, therefore, do not suffer from clogging as readily as do electronic devices, they are the preferred mode for single particle analysis approaches to quantitative latex particle agglutination assays, including immunoassays.
Optical FPAs that sense aggregate formation by the measurement of forward scattered light have been described by Masson et al. (ibid.), Masson et al. (U.S. Pat. No. 4,279,617), Cambiaso et al. (U.S. Pat. No. 4,184,849), and Cohen et al. (ibid.). Although these known systems are quantitative and sensitive, they disclose only single analyte assays, they are not aggregation rate-based methods, and they do not disclose simultaneous particle agglutination assays of multiple analytes in a single sample.
The Masson and Cambiaso systems, above, which sense forward scattered light pulses from non-aggregated particles that pass through a focused optical beam and which set electronic windows so as to ignore light pulses from aggregated particles, prefer the use of latex particles of two different sizes for agglutination, perhaps to lessen the effect that an initial distribution of multiplets (non-specifically formed without an Ag-Ab reaction occurring) may have on the assay reaction. Uzgiris et al., U.S. Pat. No. 4,191,739.
If particles of only one size are used, then the initial distribution of dimers, trimers and multimers must be taken into account when measuring the additional dimers, trimers, etc. that are created by the immunochemical process. On the other hand, if two differently sized particles are coated with the same immunochemicals needed to measure a given analyte, and are mixed together at the time the immunochemical reaction is run, then there will be no initial aggregates of the two sizes of particles. This may lessen the effect that an initial distribution of multiplets may have on the immunochemical reaction (see detailed description of the invention below).
While the use of different size particles are disclosed by Uzgiris et al., above, Masson et al., above, Cohen et al. above and Cambiaso et al., above, for single analyte testing by latex particle agglutination methods, none of these references disclose solutions to the problems of simultaneous multiple testing by latex particle agglutination. Indeed, the particle size recommendations made in these references are so incomplete that the inventions are unworkable even for single analytes. The present invention, as will be detailed below, is concerned with the specific means by which particles of differing sizes or refractive indices must be chosen and used in order to quantitatively monitor simultaneous multiple latex particle agglutination reactions.
Cambiaso et al., above, discloses a method for using a cross reactive antibody immobilized on one of the particle sizes and an antigen that reacts with only one of the antibody sites on the other size particle in an inhibition immunoassay. Although it is stated that the immobilized antigen gives specificity to the assay, and that, by choosing the correct immobilized antigen, an assay for that antigen in a patient sample can be carried out, this method specifically fails if one or more of the cross-reacting analytes is present simultaneously with other cross-reacting antigens. Therefore, the Cambiaso et al. system cannot be used for simultaneous multiple testing.
Abbott et al., U.S. Pat. No. 4,521,521, discloses a method for quantitatively measuring a single analyte in a liquid sample by measuring the rate of aggregation of analyte-bound particles. Measuring perpendicular light scatter is preferred. Abbott et al. do not teach a method of estimating multiple analytes simultaneously in the same sample, do not teach the use of different size or refractive index particles for each of multiple analytes in a single sample, and teach particles bound to analyte rather than to a ligand as in the present invention.
Abbott et al. above also disclose an analytical instrument for use with their immunoassay method. This instrument is, however, completely different in terms of concept, principle, design, electronics and operation than the optical flow particle analyzers of the present invention described below. Abbott relates to a particle size distribution measuring instrument, wherein count values for each particle size relating to the same analyte are obtained. Abbott et al. accomplish this, not by using single channel analyzers to separate pulse signals from a light detector into separate output signals or by using a peak detector means to sample peak height values of pulse signals from a detector and outputting corresponding peak height values, as are done in the instrument embodiments of the present invention, but, instead, by a counter network comprising a threshold comparator, a monostable multivibrator that generates a logic signal for each electrical pulse passing the comparator, and a counter in which is incremented the logic signals. The output of the threshold comparator equals the difference between the light detector pulse signal and a preset threshold level, and is not the output signal of the detector as is employed in the present invention. Further, the signals are representative of only a single analyte. The Abbott circuit does not separate the pulse signals from the detector, but merely triggers the comparator in an all-or-none fashion when a preset threshold level is exceeded. Because large multimers generate pulses of greater amplitude than do lower multimers or monomers, in the Abbott system the pulses from N-mer particles will exceed the thresholds of all channels and will increment all counters. These threshold circuits are clearly not single channel analyzers. Further, the threshold circuits of Abbott cannot sample peak height values, as is done by the peak detector means disclosed below, but rather are merely triggered when the signal exceeds a threshold. The signal may exceed the present threshold value, and trigger the circuit, before reaching its peak height value. In addition, the output of the threshold comparator is merely a pulse indicative of the fact that the pulse exceeded a threshold value; it provides no information as to the peak height of the signal, which peak height sampling is integral to the present FPA.
Cannon KK, JP 1207663, refers to a flow particle latex agglutination assay method and instrument for measuring multiple analytes simultaneously in a fluid sample. The patent employs particles coated with Ag or Ab specific for the Ab or Ag to be detected. Different particles may be of the same or different size. The method detects analytes by detecting light scatter in two directions, one of which is sideways, and uses an end point measurement of aggregation rather than a more advantageous rate-based assay as in the instant invention.
Thus, although particle agglutination-based assay methods that use flow or static particle analytical instruments are known, there remains an important need for a particle agglutination method capable of performing panels of in vitro laboratory tests, including immunoassays, on a simultaneous basis. That is, it would be greatly advantageous if such a simultaneous test could be performed by adding a single reagent combination to a single sample of a patient fluid sample without need to subdivide this sample, in contrast to present methods that require division of the patient sample, use of multiple reagents in multiple steps and collation of results at a later time. This need is now fulfilled by the invention described below.